I. Field of the Invention
Embodiments of this invention are directed generally to immunology and medicine. In certain embodiments the invention is directed to detection of autoreactive immune cell (B cell) that recognize the acetylcholine receptor.
II. Background
Myasthenia gravis (MG) is a human autoimmune disorder characterized by muscle weakness and fatigability. In this disease, antibodies against the acetylcholine receptor (AChR) bind to the receptor and destroy the receptor and thus interfere with the transmission of signals from nerve to muscle at the neuromuscular junction (Patrick and Lindstrom, 1973).
The acetylcholine receptor molecule is a transmembrane glycoprotein consisting of five subunits, two α, one β, one δ, with either an ε of γ subunit, organized in a barrel-staves-like structure around a central cation channel (Karlin, 1980; Changeux et al., 1984). Noda et al. (1983) described the cloning and sequence analysis of human genomic DNA encoding the α-subunit precursor of muscle acetylcholine receptor, and Schoepfer et al. (1988) reported the cloning of the α-subunit cDNA from the human cell line TE671. Human muscle AChR α-subunit exists in two forms, one of which has 25 additional amino acid residues, inserted between positions 58 and 59, that are coded by the 75 bp exon p3A (Beeson et al., 1990). The α-subunit of AChR contains both the site for acetylcholine binding and is the immunodominant region for anti-AChR immune responses. However, antibodies have been detected against all subunits of AChR.
The autoimmune response in myasthenia gravis is directed mainly towards the extracellular domain of the AChR α-subunit (amino acids 1-210), and within it, primarily towards the main immunogenic region (MIR) encompassing amino acids 61-76 (Tzartos and Lindstrom, 1980; Tzartos et al., 1987; Loutrari et al., 1992). Many antibodies to the MIR bind only to the native conformation of the α subunits because they bind to sequences that are adjacent only in the native structure.
MG is currently treated by acetylcholinesterase inhibitors and by non-specific immunosuppressive drugs that have deleterious side effects. It would be preferable to treat MG with a method that involves antigen-specific immunotherapy but leaves the overall immune response intact. One such strategy of specific therapy could involve the administration of derivatives of AChR that do not induce myasthenia but are capable of affecting the immunopathogenic antibodies. However, since the anti-AChR antibody repertoire in myasthenia gravis has been shown to be polyclonal and heterogeneous (Drachman, 1994), the regulation of the disease requires modulation of many antibody specificities.
Previous studies were directed towards modulating the anti-AChR response and EAMG by either derivatives of Torpedo AChR, e.g., the reduced and carboxymethylated derivative, RCM-AChR (Bartfeld and Fuchs, 1978), synthetic peptides corresponding to Torpedo acetylcholine receptor (Shenoy et al., 1993), specific regions of AChR (Shenoy et al., 1993; Souroujon et al., 1992; Souroujon et al., 1993), or mimotopes selected from an epitope library (Balass et al., 1993). The Torpedo RCM-AChR did not induce EAMG in rabbits and was effective in suppressing the disease. However, RCM-AChR did induce EAMG in rats. The experiments carried out with the synthetic peptides and mimotopes were only partially successful in neutralizing MG autoimmune response, probably due to the incorrect folding of the short peptides that were recognized by only a portion of the anti-AChR antibodies and ineffective tolerance to acetylcholine receptor specific B cells.
MG is currently diagnosed by testing for antibodies against AChR by radioimmunoassay wherein the antigen is crude AChR extracted from human muscle or TE671 cells. This test presents some drawbacks, namely the antigen is not readily available and, in addition, the antibody titers detected are not well correlated with disease severity.
Thus, additional methods and compositions that are both reliable and convenient diagnostic test is needed.